Laulimalide microtubule stabilizing agents

ABSTRACT

A method of inhibiting the proliferation of a hyperproliferative mammalian cell having a multiple drug resistant phenotype utilizing an amount of a laulimalide compound effective to disrupt the dynamic state of microtubule polymerization and depolymerization to arrest cell mitosis is disclosed, together with laulimalide compounds which find use in such method.

This invention was made with United States Government support underGrant Nos. 5P30CA071789-02 and 1R01CA081388-01, awarded by the NationalInstitutes of Health. The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Neoplastic diseases or cancers, characterized by the proliferation ofcells not subject to normal growth regulation, are a major cause ofdeath in humans. An estimated 1,221,800 new cases and 561,000 deaths areexpected to occur in 1999. Lung cancer remains the leading cause ofcancer-related deaths in the United States; the estimated 158,900 deathswould account for 28% of the total.

Clinical experience in chemotherapy has demonstrated that new and moreeffective cytotoxic drugs are desirable to treat these diseases. Suchexperience has also demonstrated that drugs which disrupt themicrotubule system of the cytoskeleton can be effective in inhibitingthe proliferation of neoplastic cells.

The microtubule system of eucaryotic cells is a major component of thecytoskeleton and is in a dynamic state of assembly and disassembly; thatis, heterodimers of tubulin are polymerized to form microtubules, andmicrotubules are depolymerized to their constituent components.Microtubules play a key role in the regulation of cell architecture,metabolism, and division, and the dynamic state of the microtubules iscritical to their normal function. With respect to cell division,tubulin is polymerized into microtubules that form the mitotic spindle.The microtubules are then depolymerized when the mitotic spindle's rolehas been fulfilled. Accordingly, agents which disrupt the polymerizationor depolymerization of microtubules, and thereby inhibit cell growth,comprise some of the most effective chemotherapeutic agents in clinicaluse.

Such anti-mitotic agents or poisons all kinetically inhibit the normaldynamics of microtubules. There are subtle differences between certainclasses of anti-microtubule agents based on their molecular mechanism ofaction. Colchicine binds to soluble tubulin and then is incorporatedinto a growing microtubule, vinblastine binds to the microtubule end andthereby suppresses microtubule dynamics. At high concentrations, bothcolchicine and vinblastine cause the loss of cellular microtubules.Paclitaxel (more commonly known as Taxol™) and related taxanes alsoinhibit microtubule dynamics, yet at high concentrations these agentscause an increase in polymerized tubulin in the cell and thickmicrotubule bundles are formed.

Paclitaxel was first isolated in 1971 in the bark of the Pacific yewtree (Taxus brevifolia), and was approved in 1992 by the US Food andDrug Administration for treatment of metastatic ovarian cancer and laterfor breast cancer. Paclitaxel has attracted unusually strong scientificattention, not only because of its unique anti-proliferative mechanismof action, but also because it is active against a broad range oftumors. The discovery of the effectiveness of the natural productpaclitaxel lead to the production and testing of semisynthetic congenersincluding docitaxel (Taxotere). These compounds, taxanes, are nowrecognized as a new class of anti-cancer compounds.

One drawback of paclitaxel is its extreme insolubility: Paclitaxel canbe administered effectively only in a solvent including cremophor, whichcombination can provoke severe hypersensitive immune responses. As aresult of these drawbacks, it is considered desirable to explore the useof other naturally-occurring compounds with similar modes of action.

In addition, merely having activity as an antimitotic agent does notguarantee efficacy against a tumor cell, and certainly not a tumor cellwhich exhibits a drug-resistant phenotype. Vinca alkaloids, such asvinblastine and vincristine, and taxanes are effective againstneoplastic cells and tumors, yet they lack or display reduced activityagainst drug-resistant tumors and cells. One basis for a neoplastic celldisplaying drug resistance (DR) or multiple-drug resistance (MDR) isthrough the over-expression of P-glycoprotein. Compounds which are poorsubstrates for transport of P-glycoprotein should be useful incircumventing such DR or MDR phenotypes.

Accordingly, the exhibition of the DR or MDR phenotype by many tumorcells and the clinically proven mode of action of anti-microtubuleagents against neoplastic cells necessitates the development ofanti-microtubule agents cytotoxic to non-drug resistant neoplastic cellsas well as cytotoxic to neoplastic cells with a drug resistantphenotype.

Since the discovery of the mechanism of action of paclitaxel, only threeother non-taxane chemical classes (epothilones A and B, discodermolide,and eleutherobin and related sarcodictyins A and B) have been identifiedthat posses a similar mode of action. The epothilones were isolated fromthe myxobacterium Sorangium cellulosum as a result of a large-scalescreening effort. The epothilones have generated significant interest,as they retain activity against drug-resistant cell lines. Epothiloneshave been isolated from a species of bacteria found in soil samplescollected from the banks of the Zambesi River in the Republic of SouthAfrica, and have been recently synthesized.

Discodermolide was purified from the marine sponge Discodermia dissolutaas an immunosuppressant and was screened for anti-mitotic activity onthe basis of a predictive structure-activity relationship when comparedwith other tubulin-interacting drugs. Discodermolide promotes tubulinassembly more potently than paclitaxel and it is an effective inhibitorof cell growth in paclitaxel-resistant cells.

Eleutherobin, a potent cytotoxin from the soft coral eleutherobia sp.,promotes tubulin polymerization but exhibits cross-resistance topaclitaxel-resistant cell lines. The potential therapeutic usefulness ofthese new microtubule-stabilizing compounds, and whether they willprovide advantages over the taxanes, have yet to be determined.

Accordingly, it remains desirable to identify additionalnaturally-occurring compounds with modes of action similar to thetaxanes, but which display different tissue specificity, solubility,and/or activity against drug-resistant, and particularly multiple-drugresistant, tumors and cells.

DISCLOSURE OF THE INVENTION

The present invention provides a method of inhibiting the proliferationof a hyperproliferative mammalian cell having a multiple drug resistantphenotype comprising contacting the cell with an amount of a laulimalidecompound effective to disrupt the dynamic state of microtubulepolymerization and depolymerization to arrest cell mitosis, therebyinhibiting the proliferation of the cell.

The laulimalide compounds which find use in the present invention willhave a general structure according to the following formula:

in which A and B are structural variants of Laulimalide regions A and Bas follows:

and where the epoxide ring in the formula can optionally be replacedwith a double bond, which variants preserve the ability to inhibit theproliferation of a hyperproliferative mammalian cell having a multipledrug resistant phenotype.

Specific embodiments of compounds which will be found useful in thepractice of the present invention are represented here by laulimalide:

and variants know to exist in the basic laulimalide structure, includingisolaulimalide, which differ structurally from laulimalide as follows:

Furthermore, synthetic variants of the native laulimalide structure,termed analogs, which differ structurally from laulimalide as follows,are expected to retain the ability to inhibit the proliferation of ahyperproliferative mammalian cell having a multiple drug resistantphenotype:

A further aspect of the present invention includes compositionscomprising the novel Laulimalide analog compounds.

Other aspects of the present invention will be readily apparent from thefollowing more detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood by reference to the followingdetailed description of specific embodiments when considered incombination with the drawings that form a part of this specification,wherein:

FIG. 1 is a graphic representation comparing the effects of Laulimalideand paclitaxel on micronuclei formation, in which A-10 cells weretreated for 18 hours with Laulimalide or paclitaxel, the cells werefixed and nuclei were visualized by DAPI staining;

FIG. 2 is a graphic representation comparing the effects of Laulimalideon cell cycle distribution, wherein log phase growth cultures ofMDA-MB-435 cells were treated, the cells were fixed, stained, andanalyzed on a Coulter EPICS XL-MCL flow cytometer and plotted as eventsversus propidium iodide fluorescence intensity, and in which:

FIG. 2A portrays the control cells,

FIG. 2B portrays the cells treated with 20nm Laulimalide for 9 hours,and

FIG. 2C portrays the cells treated with 20nm Laulimalide for 18 hours;and

FIG. 3 is a graphic representation comparing the effects of Laulimalideand paclitaxel on tubulin polymerization, in which the polymerization ofbovine brain tubulin was monitored, and in which:

FIG. 3A portrays the cells treated with Laulimalide, and

FIG. 3B portrays the cells treated with paclitaxel.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of inhibiting the proliferationof a hyperproliferative mammalian cell having a multiple drug resistantphenotype comprising contacting the cell with an amount of a laulimalidecompound effective to disrupt the dynamic state of microtubulepolymerization and depolymerization to arrest cell mitosis, therebyinhibiting the proliferation of the cell.

The laulimalide compounds which find use in the present invention willhave a general structure according to the following formula:

in which A and B are structural variants of Laulimalide regions A and Bas follows:

and where the epoxide ring in the formula can optionally be replacedwith a double bond, which variants preserve the ability to inhibit theproliferation of a hyperproliferative mammalian cell having a multipledrug resistant phenotype.

Specific embodiments of compounds which will be found useful in thepractice of the present invention are represented here by laulimalide:

and variants know to exist in the basic laulimalide structure, includingisolaulimalide, which differ structurally from laulimalide as follows:

Together with analog variants of laulimalide, including the followingrepresentative examples (wherein A and B represent regions wherestructural variation is permissable while retaining some measure of theactivity of the title compound laulimalide):

Certain of the laulimalide compounds of the present invention werepreviously isolated from Cacospongis mycofijiensis, a marine sponge.These include Laulimalide and isolaulimalide. Generally, the nativecompounds are isolated as follows: Lyophilized C. mycofijiensis wereground to a powder and extracted by stirring withdichloromethane/isopropanol for 24 hours at room temperature. Theextraction was repeated under identical conditions with fresh solvent,and the combined extracts were evaporated in vacuo below 40° C. Theresidue was dissolved in 90% (v/v) aqueous methanol and extracted threetimes with equal volumes of hexanes, and the hexane fractions werediscarded. The aqueous methanol phase was diluted with water to 80%(v/v) aqueous methanol and extracted three times with equal volumes oftoluene. The combined toluene fractions were evaporated in vacuo below40° C. to dryness. The residue was dissolved in a minimum amount ofdichloromethane and applied to a silica gel column equilibrated inether, and the column was eluted with ether. Active fractions werecombined and rechromatographed over silica gel using a solvent system of75% methyl-tert-butyl ether/25% hexanes (v/v) containing 1% isopropanol.

Although somewhat laborious, the structure of the laulimalides as aclass is amenable to chemical synthesis and the production of analogs,by techniques known in the art, is within the routine skill of anorganic chemist. Thus, it is anticipated that laulimalide analogs can beroutinely synthesized while preserving some measure of the activity ofthe representative members of the class of compounds and their abilityto inhibit the proliferation of a hyperproliferative mammalian cellhaving a multiple drug resistant phenotype. Thus, novel laulimalideanalogs having the following structure are also considered to be withinthe scope of the present invention:

In embodiments of the present invention, the method further comprisescontracting the cell with at least one additional anti-neoplastic agent.In further embodiments of the invention, the mammalian cell is human.

The present invention also provides a method of alleviating apathological condition caused by hyperproliferating, multiple drugresistant mammalian cells comprising administering to a subject aneffective amount of the pharmaceutical composition disclosed herein toinhibit proliferation of the cells. In further embodiments of thepresent invention, the mammalian cells are human.

In certain embodiments of the present invention, the method furthercomprises administering to the subject at least one additional therapydirected to alleviating the pathological condition. In certainembodiments of the present invention, the pathological condition ischaracterized by the formation of neoplasms. In further embodiments ofthe present invention, the neoplasms are selected from the groupconsisting of mammary, small-cell lung, non-small-cell lung, colorectal,leukemia, melanoma, pancreatic adenocarcinoma, central nervous system(CNS), ovarian, prostate, sarcoma of soft tissue or bone, head and neck,gastric which includes pancreatic and esophageal, stomach, myeloma,bladder, renal, neuroendocrine which includes thyroid and non-Hodgkin'sdisease and Hodgkin's disease neoplasms.

The novel and the previously disclosed laulimalide compounds of thepresent invention can be therapeutically employed as anti-neoplasticagents and thereby used in methods to treat neoplastic diseases. As usedherein, “neoplastic” pertains to a neoplasm, which is an abnormalgrowth, such growth occurring because of a proliferation of cells notsubject to the usual limitations of growth. As used herein,“anti-neoplastic agent” is any compound, composition, admixture,co-mixture or blend which inhibits, eliminates, retards or reverses theneoplastic phenotype of a cell.

Chemotherapy, surgery, radiation therapy, therapy with biologic responsemodifiers, and immunotherapy are currently used in the treatment ofcancer. Each mode of therapy has specific indications which are known tothose of ordinary skill in the art, and one or all may be employed in anattempt to achieve total destruction of neoplastic cells. Chemotherapyutilizing one or more laulimalides is provided by the present invention.Moreover, combination chemotherapy, chemotherapy utilizing laulimalidesin combination with other neoplastic agents, is also provided by thesubject invention as combination therapy is generally more effectivethan the use of single anti-neoplastic agents. Thus, a further aspect ofthe present invention provides compositions containing a therapeuticallyeffective amount of at least one new laulimalide compound of the presentinvention, including nontoxic addition salts thereof, which serve toprovide the above-recited therapeutic benefits. Such compositions canalso be provided together with physiologically tolerable liquid, gel orsolid carriers, diluents, adjuvants and excipients. Such carriers,diluents, adjuvants and excipients may be found in the United StatesPharmacopeia Vol. XXII and National Formulary Vol XVII, U.S.Pharmacopeia Convention, Inc., Rockville, Md. (1989), the contents ofwhich are herein incorporated by reference. Additional modes oftreatment are provided in AHFS Drug Information, 1993 ed. by theAmerican Hospital Formulary Service, pp. 522-660, the relevant contentsof which are herein incorporated by this reference.

Certain embodiments of the present invention further provide that thepharmaceutical composition used to treat neoplastic disease contains atleast one laulimalide compound and at least one additionalanti-neoplastic agent. Anti-neoplastic compounds which may be utilizedin combination with laulimalides include those provided in The MerckIndex, 11th ed. Merck & Co., Inc. (1989) pp. Ther 16-17, the contents ofwhich are hereby incorporated by reference. In a further embodiment ofthe invention, anti-neoplastic agents may be anti-metabolites which mayinclude, but are not limited to, methotrexate, 5-fluorouracil,6-mercaptopurine, cytosine arabinoside, hydroxyurea, and2-chlorodeoxyadenosine. In another embodiment of the present invention,the anti-neoplastic agents contemplated are alkylating agents which mayinclude, but are not limited to, cyclophosphamide, melphalan, busulfan,paraplatin, chlorambucil, and nitrogen mustard. In a further embodimentof the subject invention, the anti-neoplastic agents are plant-derivednatural products which may include, but are not limited to, vincristine,vinblastine, paclitaxel, and etoposide. In a further embodiment of thepresent invention, the anti-neoplastic agents contemplated areantibiotics which may include, but are not limited to, doxorubicin(adriamycin), daunorubicin, mitomycin c, and bleomycin. In a furtherembodiment of the subject invention, the anti-neoplastic agentscontemplated are hormones which may include, but are not limited to,calusterone, diomostavolone, propionate, epitiostanol, mepitiostane,testolactone, tamoxifen, polyestradiol phosphate, megesterol acetate,flutamide, nilutamide, and trilotane. In a further embodiment of thesubject invention, the anti-neoplastic agents contemplated includeenzymes which may include, but are not limited to, L-Asparaginase oraminoacridine derivatives which may include, but are not limited to,amsacrine. Additional anti-neoplastic agents include those provided inSkeel, Roland T., “Antineoplastic Drugs and Biologic Response Modifier:Classification, Use and Toxicity of Clinically Useful Agents,” Handbookof Cancer Chemotherapy (3rd ed.), Little Brown & Co. (1991), therelevant contents of which are herein incorporated by this reference.

The present laulimalide compounds and compositions can be administeredto mammals for veterinary use, such as for domestic animals, andclinical use in humans in a manner similar to other therapeutic agents.In general, the dosage required for therapeutic efficacy will varyaccording to the type of use and mode of administration, as well as theparticularized requirements of individual hosts. Ordinarily, dosageswill range from about 0.001 to 1000 mg/kg, more usually 0.01 to 10mg/kg, of the host body weight. Alternatively, dosages within theseranges can be administered by constant infusion over an extended periodof time, usually exceeding 24 hours, until the desired therapeuticbenefits have been obtained. Indeed, drug dosage, as well as route ofadministration, must be selected on the basis of relative effectiveness,relative toxicity, growth characteristics of tumor and effect oflaulimalides on cell cycle, drug pharmacokinetics, age, sex, physicalcondition of the patient, and prior treatment.

The laulimalide compounds, with or without additional anti-neoplasticagents, may be formulated into therapeutic compositions as natural orsalt forms. Pharmaceutically acceptable non-toxic salts include the baseaddition salts (formed with free carboxyl or other anionic groups) whichmay be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like. Such salts may also be formed as acidaddition salts with any free cationic groups and will generally beformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or organic acids such as acetic, oxalic, tartaric,mandelic, and the like. Additional excipients which the furtherinvention provides are those available to one of ordinary skill in theart, for example, that found in the United States Pharmacopeia Vol. XXIIand National Formulary Vol XVII, U.S. Pharmacopeia Convention, Inc.,Rockville, Md. (1989), the relevant contents of which is hereinincorporated by this reference.

The suitability of particular carriers for inclusion in a giventherapeutic composition depends on the preferred route ofadministration. For example, anti-neoplastic compositions may beformulated for oral administration. Such compositions are typicallyprepared either as liquid solution or suspensions, or in solid forms.Oral formulations usually include such normally employed additives suchas binders, fillers, carriers, preservatives, stabilizing agents,emulsifiers, buffers and excipients as, for example, pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharin, cellulose, magnesium carbonate, and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations, or powders, and typicallycontain 1%-95% of active ingredient, preferably 2%-70%.

Compositions of the present invention may also be prepared asinjectable, either as liquid solutions, suspensions, or emulsions; solidforms suitable for solution in, or suspension in, liquid prior toinjection may be prepared. Such injectables may be administeredsubcutaneously, intravenously, intraperitoneally, intramuscularly,intrathecally, or intrapleurally. The active ingredient or ingredientsare often mixed with diluents or excipients which are physiologicallytolerable and compatible with the active ingredient(s). Suitablediluents and excipients are, for example, water, saline, dextrose,glycerol, or the like, and combinations thereof. In addition, ifdesired, the compositions may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, stabilizing or pHbuffering agents.

The invention further provides methods for using laulimalide compoundsencompassed by the genus structure to inhibit the proliferation ofmammalian cells by contacting these cells with a laulimalide compound inan amount sufficient to inhibit the proliferation of the mammalian cell.A preferred embodiment is a method to inhibit the proliferation ofhyperproliferative mammalian cells. For purposes of this invention,“hyperproliferative mammalian cells” are mammalian cells which are notsubject to the characteristic limitations of growth, e.g., loss ofgrowth control and insensitivity to normal programmed cell death(apoptosis). A further preferred embodiment is when the mammalian cellis human. The invention further provides contacting the mammalian cellwith at least one laulimalide compound and at least one additionalanti-neoplastic agent. The types of anti-neoplastic agents contemplatedare the same as those disclosed hereinabove.

The invention further provides methods for using laulimalide compoundsencompassed by the genus structure to inhibit the proliferation ofhyperproliferative cells with drug-resistant phenotypes, including thosewith multiple drug-resistant phenotypes, by contacting said cell with alaulimalide compound in an amount sufficient to inhibit theproliferation of a hyperproliferative mammalian cell. A preferredembodiment is when the mammalian cell is human. The invention furtherprovides contacting the mammalian cell with a laulimalide compound andat least one additional anti-neoplastic agent. The types ofanti-neoplastic agents contemplated are the same as those disclosedhereinabove.

The invention further provides a method for alleviating pathologicalconditions caused by hyperproliferating mammalian cells, for example,neoplasia, by administering to a subject an effective amount of apharmaceutical composition provided hereinabove to inhibit theproliferation of the hyperproliferating cells. As used herein“pathological condition” refers to any pathology arising from theproliferation of mammalian cells that are not subject to the normallimitations of cell growth. Such proliferation of cells may be due toneoplasms, including, but not limited to the following neoplasms:mammary, small-cell lung, non-small-cell lung, colorectal, leukemia,melanoma, central nervous system (CNS), ovarian, prostate, sarcoma ofsoft tissue or bone, head and neck, gastric which includes pancreaticand esophageal, stomach, myeloma, bladder, renal, neuroendocrine whichincludes thyroid and lymphoma, non-Hodgkin's and Hodgkin's. In a furtherembodiment of the invention, the neoplastic cells are human. The presentinvention further provides methods of alleviating such pathologicalconditions utilizing laulimalide in combination with other therapies, aswell as other anti-neoplastic agents. Such therapies and theirappropriateness for different neoplasia may be found in CancerPrinciples and Practice of Oncology, 4th ed., Editors DeVita, V.,Hellman, S., and Rosenberg., S., Lippincott Co. (1993), the contents ofwhich are herein incorporated by reference.

In the present disclosure, laulimalide compounds are shown to potentlystabilize the microtubule structure in cultured cells. In addition, andin contrast with the Vinca alkaloids and paclitaxel, laulimalidecompounds appear to be a poor substrate for the drug-efflux pumpP-glycoprotein.

The following examples serve to illustrate certain embodiments andaspects of the present invention and are not to be construed as limitingthe scope thereof.

Experimental

In the experimental disclosure which follows, all weights are given ingrams (g), milligrams (mg), micrograms (μg), nanograms (ng), orpicograms (pg), all amounts are given in moles (mol), millimoles (mmol),micromoles (μmol), nanomoles (nmol), picomoles (pmol), or femtomoles(fmol), all concentrations are given as percent by volume (%),proportion by volume (v:v), molar (M), millimolar (mM), micromolar (μM),nanomolar (nM), picomolar (pM), femtomolar (fM), or normal (N), allvolumes are given in liters (L), milliliters (mL), or microliters (μL),and linear measurements are given in millimeters (mm), or nanometers(nm) unless otherwise indicated.

The following examples demonstrate the practice of the present inventionin isolating, purifying and employing laulimalide microtubulestabilizing agents for inhibiting the proliferation of ahyperproliferative mammalian cell having a multiple drug resistantphenotype.

Reagents

4,6-Diamidino-2-phenylindole (DAPI), sulforhodamine B (SRB), antibodiesagainst β-tubulin, and Basal Medium Eagle containing Earle's salts (BME)were obtained from the Sigma Chemical Company (St. Louis, Mo.).Richter's medium was obtained from BioWhittaker (Walkersville, Md.) andFetal Bovine Serum (FBS) was obtained from Hyclone Laboratories (Logan,Utah).

Cell Lines

The A-10 rat aortic smooth muscle and SK-OV-3 human ovarian carcinomacell lines were obtained from the American Type Culture Collection(Manassas, Va.) and were cultured in BME containing 10% FBS and 50 μg/mLgentamycin sulfate. A sub-line of SK-OV-3 selected for resistance tovinblastine (SKVLB-1) was provided by Dr. Victor Ling (British ColumbiaCancer Center, Vancouver, British Columbia) and was maintained in BMEcontaining 10% FBS and 50 μg/mL gentamycin sulfate. The MDA-MB-435 humanmammary adenocarcinoma cell line was obtained from Dr. Mai Higazi(Georgetown University, Washington, D.C.), and was maintained inRichters medium containing 10% FBS and 50 μg/mL gentamycin sulfate.Vinblastine was added to a final concentration of 1 μg/mL to SKVLB-1cells 24 hours after passage to maintain selection pressure forP-glycoprotein-overexpressing cells.

Inhibition of Cell Proliferation

The IC₅₀ for inhibition of cell proliferation was determined bymeasuring cell-associated protein after drug treatment using thesulforhodamine B assay.

Immunofluorescence Assays

A-10 cells were grown to 70-85% confluence on glass coverslips in BMEsupplemented with 10% FBS. Drug compounds in PBS were added to theindicated final concentrations and cells were incubated for anadditional 24 hours.

For the staining of microtubules and intermediate filaments, the cellswere fixed with cold methanol for 5 minutes, blocked for 20 minutes withPBS containing 10% calf serum to block nonspecific binding sites, andincubated at 37° C. for 90 min with monoclonal anti-β-tubulin at thedilutions recommended by the manufacturer. Bound primary antibodies weresubsequently visualized by a one hour incubation withfluorescein(FITC)-conjugated sheep antimouse IgG (F-3008; Sigma). Thecoverslips were washed, stained with 0.1 μg/mL DAPI for 10 minutes,mounted on microscope slides and the fluorescence patterns were examinedand photographed using a Zeiss Axioplan microscope equipped withepifluorescence optics for fluorescein and DAPI.

Effects of Laulimalide, Isolaulimalide, and Paclitaxel on CellularMicrotubules.

Strong paclitaxel-like microtubule-stabilizing activity was found in thecrude lipophilic extract from the marine sponge C. mycofijiensis. Theextract was cytotoxic, and after treatment the only cell remnants thatremained where thick, short microtubule bundles. Bioassay-directedpurification of the extract yielded the microtubule active-compoundslaulimalide and isolaulimalide, as well as the microfilament disrupterlatrunuculin A. Studies with the purified compounds show that bothlaulimalide and isolaulimalide caused dramatic reorganization ofcellular microtubules.

A-10 cells were treated with laulimalide, isolaulimalide, or paclitaxelfor 18 hours and the morphological effects on microtubules examined byindirect immunofluorescence techniques. The control cells exhibitednormal microtubules arrays with filamentous microtubules radiating fromthe microtubule organizing center to the cell periphery. Treatment ofthe cells with laulimalide disrupted the normal microtubule array; themicrotubules were more numerous and appeared to occupy more of thecytoplasm. A 2 μM laulimalide exhibited bundles of short tufts ofmicrotubules that were prevalent in the cell periphery and seemed to beindependent of nucleation from microtubule organizing centers.Isolaulimalide at concentrations between 2-20 μM caused an increase inthe density of cellular microtubules, but no microtubule bundles werepresent. Paclitaxel at concentrations between 1-20 μM initiated theformation of a highly organized array of microtubules, some of whichformed thick microtubule bundles. Long thick microtubule bundles oftensurrounded the nucleus. The extensive long microtubule hoops and bundlesthat formed after treatment with 2 μM paclitaxel were not seen withlaulimalide. Cellular microtubules stabilized with paclitaxel,laulimalide or isolaulimalide were resistant to vinblastine-induceddepolymerization.

Effects of Laulimalide and Isolaulimalide on Nuclear Structure.

A characteristic of both A-10 and SK-OV-3 cells treated with a widerange of concentrations of laulimalide and isolaulimalide was theformation of multiple micronuclei. The effects of laulimalide on nuclearstructure are visible: The normal rounded shape of the nucleus, which isdevoid of microtubules, can be detected, whereas in laulimalide-treatedcells this distinct microtubule-free area containing the discretecentral nucleus was lost and only vesicle-like areas devoid ofmicrotubules remained. Nuclear staining of control cells revealed acentral compact nucleus, whereas laulimalide-treated cells exhibited adramatic breakdown of the nucleus into micronuclei. Similar nuclearchanges occurred with isolaulimalide. Paclitaxel also initiated theformation of micronuclei, as has been reported by others. Analysis ofthe incidence of micronuclei formation showed that a higher percentageof laulimalide-treated cells contained micronuclei than cells treatedwith the same concentrations of paclitaxel.

The data in FIG. 1 demonstrate that 0.02-2 μM laulimalide causedmicronuclei formation in approximately 60% of A-10 cells. Paclitaxel atthe same concentrations caused approximately 40% of the cells to exhibitthis abnormal restructuring of the nucleus. At all concentrationstested, laulimalide caused a higher percentage of cells to exhibitmicronuclei when compared with paclitaxel-treated cells. The differencebetween the two drugs was statistically significant at all fiveconcentrations tested (P<0.0001).

Effects of Laulimalide on Cell Cycle Progression and Mitotic Spindles.

A common characteristic of anti-microtubule agents is their ability toinitiate mitotic arrest. Flow cytometric analysis revealed thatlaulimalide-induced cell cycle arrest in G₂-M in MDMB-435 breastcarcinoma cells within nine hours of treatment (FIG. 2). This isconsistent with the effects of other antimicrotubule agents, wheredisruption of microtubule dynamics prevents normal mitotic progressionand leads to mitotic arrest. Abnormal mitotic spindles were seen in bothA-10 cells and SK-OV-3 cells after treatment with laulimalide andisolaulimalide. The mitotic cells were rounded, and the spindles formeda circular pattern with spindle microtubules radiating outward from amicrotubule-free core in the center of the cell. In the mitotic cells,the nuclear membranes were not apparent, and the chromatin was condensedand aligned in a circular pattern. Abnormal mitotic spindles werevisible in paclitaxel-treated cells and were typically tri- ortetra-polar and did not exhibit the morphology seen with thelaulimalides.

Effects of Laulimalide, Isolaulimalide, and Paclitaxel on CellProliferation of Drug-Sensitive and Multidrug-Resistant Cell Lines.

The early literature on laulimalide reported that it was a cytotoxin,but without specifying a mechanism. Experiments were conducted todetermine the IC₅₀ values for laulimalide and isolaulimalide in twodrug-sensitive cell lines, MDA-MB-435 and SK-OV-3, and in amultidrug-resistant cell line, SKVLB-1. Cells were treated with varyingconcentrations of the drugs for 48 hours, and cell-associated proteinwas determined using the SRB assay. The IC₅₀ for each compound wascalculated for each cell line.

Laulimalide was found to be a potent inhibitor of cell proliferationwith IC₅₀ values between 5-12 nM (Table 1).

TABLE 1 Cell Line Laulimalide Isolaulimalide Paclitaxel MDA-MB-435 5.74± 0.58 1,970 ± 97  1.02 ± 0.25 SK-OV-3 11.53 ± 0.53  2,570 ± 290 1.71 ±1.07 SKVLB 1,210 ± 490     2,650 ± 1,384 >100,000 Resistance Factor 1051.03  >58,480

The values represent the means of three (laulimalide, isolaulimalide) orfour (paclitaxel) experiments±SD

Isolaulimalide is less potent with IC₅₀ values in the low μM range. Bothlaulimalide and isolaulimalide inhibited the proliferation of theSKVLB-1 cell line that overexpresses the drug efflux pumpP-glycoprotein. The resistance factors between the parental,drug-sensitive line (SK-OV-3) and the drug-resistant cell line (SKVLB-1)were 105 and 1.03 for laulimalide and isolaulimalide, respectively(Table 1). Greater than 70% inhibition of the SKVLB-1 cell line was notachieved with concentrations of paclitaxel up to 100 μM. Laulimalide andisolaulimalide are significantly more effective against the SKVLB-1 cellline than paclitaxel. These data confirm that these new agents are poorsubstrates for transport by P-glycoprotein.

The IC₅₀ for inhibition of proliferation was also determined in the A-10cell line, a nontransformed line that was used to show the effects ofmicrotubule-stabilizing agents on cellular structures. The IC₅₀ forlaulimalide was 51 μM and for paclitaxel was 40 μM. The nontransformedA-10 cell line was much less sensitive to the inhibitory effects of bothpaclitaxel and laulimalide, illustrating the need to use micromolarconcentrations in these cells to induce abnormal microtubules.

Initiation of Apoptosis by Laulimalide.

The ultimate mechanism of action of many cytotoxic cancerchemotherapeutic agents is the initiation of pathways of gene andprotein expression leading to apoptosis. Antimicrotubule drugs includingpaclitaxel, vinblastine, and cryptophycin 1 initiate apoptosis both invitro and in vivo. The flow cytometry data (FIG. 2) show a doubling ofthe subdiploid peak at 18 hours (FIG. 2C), suggesting the initiation ofapoptosis. The loss of cellular DNA is detected by the appearance of thesubdiploid peak when apoptotic cells are analyzed by flow cytometry.Studies were undertaken to determine whether laulimalide initiates agene-driven program of cellular suicide.

During apoptosis, specific cysteine proteases called the caspases areactivated. Activation of the caspase cascade leads to the proteolyticdegradation of specific cellular proteins. The activation of caspase 3and the proteolysis of the DNA repair enzyme PARP, a downstreamsubstrate of caspase 3, were examined in cell lysates fromlaulimalide-treated cells. Activation of caspase 3 leads to the loss ofthe 32 kDa proenzyme and the formation of the activation products p17and p12. Analysis of immunoblot data from cell lysates shows theformation of the p17 activation product at 24, 42 and 48 hours afterlaulimalide treatment. The loss of the p32 proenzyme is seen at 48hours. The specific proteolysis of PARP by caspase 3 leads to theformation of two products, an 89 kDa COOH-terminal fragment and a 24 kDaN-terminal fragment. The proteolysis of PARP and the appearance of the89 kDa degradation product coincided with the activation of caspase 3.Caspase 3 was activated and PARP proteolytically cleaved in cell lysatesfrom cells treated with laulimalide for 42 and 48 hours. These data areconsistent with laulimalide-induced apoptotic cell death.

Effects of Laulimalide on Tubulin Polymerization in Vitro.

One characteristic of the microtubule-stabilizing agents paclitaxel,discoldermolide, epothilones A and B, and eleutherobin is the ability ofthese agents to initiate the polymerization of tubulin in the absence ofpolymerization promoters, such as glycerol. Studies were undertaken todetermine the effects of laulimalide on tubulin polymerization. Bothisolaulimalide and laulimalide stimulated tubulin polymerization. FIG.3A shows the effects of laulimalide on tubulin polymerization, and theaccompanying FIG. 3B shows the effects of paclitaxel. Comparisonsbetween the effects of laulimalide and paclitaxel on tubulinpolymerization (FIGS. 3, A and B) show that, at low micromolarconcentrations, paclitaxel is more potent than laulimalide (see alsoTable 2).

TABLE 2 Drug EC₅₀ Laulimalide 4.32 ± 0.4  Paclitaxel 1.44 ± 0.06

At low micromolar concentrations more tubulin polymer was formed in thepresence of paclitaxel, and the rate of polymerization was faster thanwas seen with equivalent concentrations of laulimalide.

A very different relationship was seen when comparing the 20 μMconcentrations of laulimalide and paclitaxel. The 20 μM concentration oflaulimalide was more effective at stimulating the formation of tubulinpolymer than was the 20 μM concentration of paclitaxel (FIGS. 3A and3B). Laulimalide promoted the polymerization of approximately 30% moretubulin polymer than was formed in the presence of paclitaxel, and thekinetics of tubulin formation were twice as fast as the rate measured inthe presence of 20 μM paclitaxel.

The tubulin polymers that formed in the presence of laulimalide wereinsensitive to cold and CaCl₂-induced depolymerization. Neitherpaclitaxel nor laulimalide promoted the polymerization of tubulin at 0°C., and laulimalide stabilized tubulin polymer formed in the presence ofGTP.

Samples of the tubulin polymer formed were examined by electronmicroscopy to determine whether the increase in turbidity measuredduring the polymerization experiments was due to the formation ofmicrotubule-like polymers or the formation of other strictures. Underhigh magnification (×63,000) the tubulin polymers formed by paclitaxeland laulimalide were indistinguishable. Both agents formed structuresresembling tubules with evidence of longitudinal symmetry. Examinationof tubulin polymers formed in the presence of themicrotubule-stabilizing agents at lower magnification (×4,000) showedthat the polymers formed with laulimalide were very long structures withrounded curves. The polymers formed with paclitaxel were notably shorterand exhibited no rounded curves, but instead formed linear structureswith angular branches.

Laulimalide and isolaulimalide were first isolated on the basis of theircytotoxicity, however, the mechanism of action was not elucidated. Theseagents have now been shown to be paclitaxel-like stabilizers ofmicrotubules that cause alterations of both inerphase and mitoticmicrotubules. Laulimalide is a potent inhibitor of cell proliferationand initiates mitotic arrest, micronuclei formation, and ultimatelyapoptosis. These compounds are superior to paclitaxel in their abilityto circumvent P-glycoprotein-mediated drug resistance. The laulimalidesrepresent a new class of paclitaxel-like microtubule-stabilizing agentswith properties that may provide advantages over the taxanes.

Laulimalide and isolaulimalide are chemically related compounds, withisolaulimalide being a decomposition product of laulimalide. Thedifference between these two compounds is in the size and attachmentpoints of the oxygen-containing ring within the top portion of themolecules. Laulimalide contains a three-membered expocide ring involvingcarbons C-16 and C-17, whereas isolaulimalide contains a five-memberedtetrahyddrofuran ring linking carbon-C-17 with side chain carbon C-20.This slight chemical difference between laulimalide and isolaulimalideresults in a difference in potency of greater than two orders ofmagnitude in their ability to inhibit cell proliferation. Furthermore,this site also seems to be crucial for recognition by the multidrugefflux pump P-glycoprotein. Laulimalide had a resistance factor of 105when comparing the IC₅₀ values in SK-OV-3 cells and SKVLB-1 cells,whereas isolaulimalide was equally sensitive in both cell lines. Thesedata demonstrate that the epoxide moiety of laulimalide is critical forits interaction with both tubulin and P-glycoprotein.

Among the five groups of known antimicrotubule agents havingpaclitaxel-like microtubule-stabilizing properties, laulimalide mostclosely resembles the epothilones. Although the ring size of laulimalideis two carbons larger (18-membered versus 16-membered), both contain asimilar structural motif that incorporates the epoxide ring, anunsaturated side chain bearing a methylated heterocyclic ring, and theester of the macrocyclic lactone ring. This similarity seems totranslate to similar activities. Both stabilize microtubules, and likelaulimalide, the epothilones are poor substrates forP-glycoprotein-mediated transport.

The cellular effects of laulimalide are similar to, but distinct from,the cellular effects of paclitaxel. The increase in the density ofcellular microtubules observed at low concentrations of laulimalideclosely resembled the changes induced by paclitaxel at the sameconcentrations. However, at concentrations above 2 μM, the effectsdiverged and laulimalide initiated short thick bundles of microtubulesthat were more prevalent in the cell periphery and appeared to form frommany nucleation centers. These effects are consistent with the greaterefficacy of 20 μM laulimalide in promoting the polymerization ofpurified tubulin. In contrast, paclitaxel-induced microtubule bundleswere long and thick and aligned in the central areas of the cellssurrounding the nucleus, consistent with nucleation from one or twocenters. Long microtubule bundles were not seen in cells treated with awide range of laulimalide concentrations. These data demonstrate that incells, laulimalide was not as effective as paclitaxel at elongation ofmicrotubules, but was more effective at stimulating the formation ofmicrotubules from multiple nucleation centers, resulting in shortermicrotubule bundles in the periphery rather than the long microtubulebundles surrounding the nucleus.

The microtubules that form the mitotic spindle are highly dynamicstructures and are more sensitive to disruption by antimicrotubuleagents than are the less dynamic interphase microtubules. Agents thattarget microtubules disrupt mitotic spindle dynamics, thereby preventingnormal mitosis, leading to mitotic arrest. Mitotic spindles formed inthe presence of laulimalide were abnormal and formed unique starburstarrays in contrast to the short thickened tri- and tetra-polar spindlesformed in the presence of paclitaxel. Laulimalide-treated mitotic cellsexhibited chromatin condensation, loss of the nuclear envelope andabnormal chromatin alignment. The aberrant mitotic spindles wereassociated with circular chromatin arrays, suggesting that themicrotubules were coordinating a specific, but abnormal structuring ofthe chromatin. Disruption of the mitotic apparatus by laulimalidetreatment lead to mitotic arrest, followed by the initiation ofapoptosis, as determined by the increase in cells in G₂-M and theactivation of the caspase cascade.

Cells treated with laulimalide exhibited vesicle-like structures in thecentral region of the cell. DAPI staining revealed that these structureswere composed of DNA and that laulimalide initiated the formation ofmultiple micronuclei. In A-10 cells, micronuclei were found in themajority of cells with treatments of 20 nM-2 μM laulimalide. Micronucleiformations occur as a consequence of treatment with either paclitaxel orepothilones and are thought to be the result of abnormal mitosis leadingto abnormal chromosome segregation.

One characteristic of the paclitaxel-like microtubule-stabilizing agentsis their ability to promote the polymerization of tubulin. Normallytubulin will not polymerize without tubulin promoters; however,laulimalide, like paclitaxel, stimulated the polymerization of tubulinin the absence of microtubule-associated proteins and glycerol. The rateand extent of polymerization in the presence of laulimalide wasdependent on concentration and differed significantly from paclitaxel,suggesting that there are differences in their mechanisms of action.Further experimentation in the presence of difference tubulin andlaulimalide concentrations and in the presence and absence ofmicrotubule-associated proteins will allow more comprehensivecomparisons of the tubulin polymerizing effects of laulimalide ascompared with paclitaxel, discodermolide, and the epothilones.

The tubulin polymers formed in the presence of laulimalide wereindistinguishable from paclitaxel-induced polymers under highmagnification, but were noticeably longer than the paclitaxel-inducedpolymers when visualized under lower magnification. Under theseconditions, laulimalide seems to promote polymer-elongating activitymore readily than paclitaxel. These in vitro effects with purifiedtubulin differ from the effects in cells where paclitaxel promoteslonger microtubules, whereas laulimalide promotes short microtubules.The initial results demonstrate that there are intriguing differences inthe mechanisms of action of laulimalide and paclitaxel.

The clinical success of the taxanes in treating a wide range of tumorshas lead to the search for new agents with a similar mechanism ofaction. The recent discovery of the beneficial activities of the classof laulimalides provides new drugs which are expected to prove useful totreat cancer.

All patents and patent applications cited in this specification arehereby incorporated by reference as if they had been specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be apparent to those of ordinary skill in the artin light of the disclosure that certain changes and modifications may bemade thereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. A method of inhibiting the proliferation of ahyperproliferative mammalian cell having a multiple drug resistantphenotype comprising contacting the cell with an amount of a laulimalidecompound effective to disrupt the dynamic state of microtubulepolymerization and depolymerization to arrest cell mitosis, therebyinhibiting the proliferation of the cell.
 2. The method according toclaim 1 wherein the laulimalide compound is a compound according to theformula:

wherein the epoxide ring in the formula can optionally be replaced witha double bond, and wherein A is a structure in accordance with theformula:

 B is a structure in accordance with the formula:

 with the proviso that at least one of regions A or B includes astructural modification which distinguishes the structure from thatdepicted in the formula, while preserving the ability of the compound todisrupt the dynamic state of microtubule polymerization anddepolymerization to arrest cell mitosis, thereby inhibiting theproliferation of the cell.
 3. The method according to claim 1 whereinthe laulimalide compound is laulimalide having the following structure:


4. The method according to claim 1 wherein the laulimalide compound isisolaulimalide having the following structure: